Peptidoglycan-Free Bacterial Ghosts Confer Enhanced Protection against Yersinia pestis Infection

To develop a modern plague vaccine, we used hypo-endotoxic Yersinia pestis bacterial ghosts (BGs) with combinations of genes encoding the bacteriophage ɸX174 lysis-mediating protein E and/or holin-endolysin systems from λ or L-413C phages. Expression of the protein E gene resulted in the BGs that retained the shape of the original bacterium. Co-expression of this gene with genes coding for holin-endolysin system of the phage L-413C caused formation of structures resembling collapsed sacs. Such structures, which have lost their rigidity, were also formed as a result of the expression of only the L-413C holin-endolysin genes. A similar holin-endolysin system from phage λ containing mutated holin gene S and intact genes R-Rz coding for the endolysins caused generation of mixtures of BGs that had (i) practically preserved and (ii) completely lost their original rigidity. The addition of protein E to the work of this system shifted the equilibrium in the mixture towards the collapsed sacs. The collapse of the structure of BGs can be explained by endolysis of peptidoglycan sacculi. Immunizations of laboratory animals with the variants of BGs followed by infection with a wild-type Y. pestis strain showed that bacterial envelopes protected only cavies. BGs with maximally hydrolyzed peptidoglycan had a greater protectivity compared to BGs with a preserved peptidoglycan skeleton.

IDENTIFICATION OF CORRELATES OF PROTECTION FROM YERSINIA PESTIS ON A MOUSE MODEL AND ASSESSMENT OF THE POSSIBILITY OF USING THEM AS MARKERS OF VACCINATION EFFICIENCY IN HUMANS

In conditions when the assessment of changes in the incidence rate cannot be used as an indicator of the effectiveness of a live plague vaccine, there is a real need to search for other, in particular, immunological correlates of the vaccine's protection. Modern concepts of the patho- and immunogenesis of plague make it possible to narrow the search for possible correlates of protection, focusing on the assessment of cellular factors of the immune response. The aim of this work is to identify the immunological correlates of protection against plague in mice immunized with Yersinia pestis EV NIIEG, and to assess the dynamics of selected markers of immunological effectiveness of vaccination in people vaccinated against plague. Experimental model - BALB / c mice, 40 individuals in each group were immunized with Y. pestis EV at doses of 2 × 102, 1 × 103, 5 × 103, 2.5 × 104 CFU, and on the 21st day they were infected with Y. pestis 231 at a dose of 400 LD50. Control group - intact animals. Immunogenicity was determined by ImD50 and calculated by the Kerber method. Volunteers - 20 people who were first vaccinated with the live plague vaccine and 20 people who were not vaccinated against the plague (comparison group). The production of cytokines in the blood was determined on an enzyme-linked immunosorbent analyzer "LAZURIT" (Dynex Technologies, USA): in mice before infection with Y. pestis 231 on the 14th and 21st days after vaccination; in humans - before vaccination, 1, 6 and 12 months after vaccination. We used commercial kits in accordance with the instructions for their use. The immunized mice showed a significant increase (2.2 times) in the induced IFN-γ production and a moderate increase in the concentration of TNF-α, IL-10 and IL-17A on the 14th day of immunogenesis. A high correlation was found between the survival rate of animals and the level of antigen- / mitogen-induced production of IFN-γ (r = 0.94, p = 0.039), both on the 14th and 21st days, as well as a noticeable relationship with the level of production of IL-10 and IL-17A on the 14th day of immunogenesis. In volunteers one month after inoculation, an increase in the indicators of mitogen-induced production of all detectable cytokines was noted, but the levels of IFN-γ, TNF-α, IL-10, IL-17A significantly increased by the 6th month of observation (p <0.05), although only for IFN-γ and IL-17A, the induced production of these cytokines remained at a sufficiently high level up to a year after inoculation. Thus, IFN-γ and IL-17A can be considered as possible informative correlates of protection of mice from Y. pestis on days 14 and 21, considering the increase in the induced production of these cytokines as adequate markers of the protective efficacy of immunization, and the assessment of the dynamics of these parameters in volunteers vaccinated with the plague live vaccine, an increase in the levels of IFN-γ and IL-17A can be considered a favorable prognostic marker of the immunological efficacy of the vaccine in the period from the 6th to the 12th month of observation.

Peptidoglycan-Free Bacterial Ghosts Confer Enhanced Protection against Yersinia Pestis Infection

To develop a modern plague vaccine, we used hypo-endotoxic Yersinia pestis bacterial ghosts (BGs) with combinations of genes encoding the bacteriophage ɸX174 lysis-mediating protein E and/or holin-endolysin systems from &lambda; or L-413C phages. Expression of the protein E gene resulted in the BGs that retained the shape of the original bacterium. Co-expression of this gene with genes coding for holin-endolysin system of the phage L-413C caused formation of structures resembling collapsed sacs. Such structures, which have lost their rigidity, were also formed as a result of the expression of only the L-413C holin-endolysin genes. Similar holin-endolysin system from phage &lambda; containing mutated holin gene S and intact genes R-Rz coding for the endolysins caused generation of mixtures of BGs that had (i) practically preserved and (ii) completely lost their original rigidity. The addition of protein E to the work of this system shifted the equilibrium in the mixture towards the collapsed sacs. The collapse of the structure of BGs can be explained by endolysis of peptidoglycan sacculi. Immunizations of laboratory animals with the variants of BGs followed by infection with a wild-type Y. pestis strain showed that bacterial envelopes protected only cavies. BGs with peptidoglycan maximally hydrolyzed had a greater protectivity compared to BGs with preserved peptidoglycan skeleton.

Efficiency of human plague vaccination in tuvinian natural plague focus. Message 2: dynamics of immune status indicators after revaccination

Relevance. In Russia, the live plague vaccine (LPV) is used for specific prophylaxis of plague. Immunological monitoring of humans vaccinated by LPV in order to search for informative diagnostic markers, as well as to improve the tactics of epidemiological surveillance of plague enzootic territories is an urgent area of research.The aim is to assess the parameters of cellular and humoral immunity in humans revaccinated by LPV who permanently reside on territory of the Tuvinian natural plague focus.Materials and methods. The study involved 76 volunteers from the Republic of Tuva, revaccinated by LPV. Blood sampling was performed before vaccination and 1, 3, and 6 months after revaccination. The study included the determination of cytokine production (IFN-γ, IL-4, TNF-α), specific antibodies, immunoglobulins (IgM, IgG, IgA and IgE) and lymphocyte subpopulation composition (CD3, CD4, CD8, CD16, CD19, immunoregulatory index).Results. A decrease in IgG and IgM and an increase in IgA were found after vaccination with LPV and their increase after revaccination. Correlation relationships were revealed between immunoglobulins, B cells and IL-4. Revaccination leads to an increase seroconversion. The activation of humoral immunity in humans vaccinated against plague is also evidenced by dynamics of changes in the subpopulation composition: an increase in B-lymphocytes and natural killer cells, a decrease in T-helpers and immunoregulatory index, and cellular immunity stimulation is an increase in spontaneous and induced production of pro- and anti-inflammatory cytokines.Conclusion. It has been shown that LPV is capable of causing the body’s immune restructuring and activating the cellular and humoral mechanisms of immunological protection. For a complete understanding of the development and preservation of antiplague immunity, it is necessary to continue the annual immunological monitoring of the population living on the territory of the Tuvinian natural plague focus, using additional modern research methods.

Impact of Toll-Like Receptor-Specific Agonists on the Host Immune Response to the Yersinia pestis Plague rF1V Vaccine

Relatively recent advances in plague vaccinology have produced the recombinant fusion protein F1-V plague vaccine. This vaccine has been shown to readily protect mice from both bubonic and pneumonic plague. The protection afforded by this vaccine is solely based upon the immune response elicited by the F1 or V epitopes expressed on the F1-V fusion protein. Accordingly, questions remain surrounding its efficacy against infection with non-encapsulated (F1-negative) strains. In an attempt to further optimize the F1-V elicited immune response and address efficacy concerns, we examined the inclusion of multiple toll-like receptor agonists into vaccine regimens. We examined the resulting immune responses and also any protection afforded to mice that were exposed to aerosolized Yersinia pestis. Our data demonstrate that it is possible to further augment the F1-V vaccine strategy in order to optimize and augment vaccine efficacy.

Comparative Analysis of the Immunogenic Activity of the Plague Vaccine Depending on the Growing Medium

The aim was to carry out a comparative analysis of the immunogenic activity of the live plague vaccine obtained on various nutrient media.Materials and methods. The subject of the study was the blood of outbred white mice immunized with a series of live plague vaccine based on Yersinia pestis EV NIIEG strain, produced using experimental and regulated nutrient media. The immunogenic activity of vaccines was studied through flow cytometry. The intensity of antigen-reactivity of lymphocytes was determined in cell tests in vitro, analyzing the early activation marker CD25+ . For the specific activation of lymphocytes, a complex of water-soluble antigens of the plague microbe was used. To identify the interdependence between the presence of protective anti-plague immunity and the level of CD 25+ expression intensity, the ED50 of the series under study was determined by the standard method.Results and discussion. A comparative analysis of the immunogenic activity of the live plague vaccine obtained on the experimental nutrient medium with the vaccine produced on Hottinger’s agar has been performed. When animals were immunized with doses of 4·103 , 2·104 and 1·105 live microbial cells (regulated doses), the highest level of expression of CD25 marker by lymphocytes was on the day 14, with a subsequent decrease on the day 21 after vaccination. When determining immunogenicity using the conventional method, a high degree of direct correlation between the number of surviving animals and an increase in the level of lymphocytes expressing markers of early activation has been established. Comparison has revealed the general pattern: when the lowest immunizing dose (8·102 ) was administered, activation of early immunity markers was not observed. In case of immunization with higher doses on days 7, 14 and 21, a proportional increase in the number of CD25-positive lymphocytes after stimulation with a specific antigen under in vitro conditions is detected in the blood of biomodels.

Improvement of Microbial Cell Concentration Technology in the Production of Live Plague Vaccine in the Form of Orodispersible Tablets

The aim of the study was to improve the procedure for the Yersinia pestis EV strain cell concentration using the system for tangential flow microfiltration with the ASF-020 filter support unit.Materials and methods. The study used the vaccine Y. pestis EV strain derived from NIIEG cell line. Submerged cultivation of the native culture was performed using BIOR-0.25 reactor with automated control system. Microbial suspension concentrate was produced through microfiltration applying (Adaptive filtration system) AFS-009 and AFS-020 installations. The content of live microbial cells was determined by cytorefractometry. Assessment of the resistance of Y. pestis EV strain cells to technological factors was performed by photometric registration of changes in the optical density of bacterial suspension during the lytic response of cells to sodium dodecyl sulfate. Physical-chemical and immunobiological properties of the dry live plague vaccine were determined in accordance with the pharmacopoeial item.3.3.1.0021.15 of the State Pharmacopoeia of the Russian Federation, 14th edition.Results and discussion. The design features of the equipment introduced made it possible to carry out membrane filtration of microbial suspension, using BIOR-0.25 reactor as an intermediate storage unit, thereby excluding three technological stages. The total concentration of microbes in the suspension obtained by routine and improved methods was more than 150 billion microbial cells per ml. A comparative study of the effect of various hydrodynamic regimes in the working cavities of AFS-009 and AFS-020 filter units did not significantly affect the morphometric properties and resistance of microbial cultures to extreme (technological) factors. Based on the experimental data, the mass balance of the membrane filtration process has been determined. The optimized technology gave 0.13 liter yield of concentrate from 1 liter of native culture, and the process duration was reduced to 5 hours, the yield of the finished product in one production cycle was increased by 3 times. Thus, the process of concentrating Y. pestis EV strain cells during the production of the tablet form of live plague vaccine has been enhanced. A comparative study of the morphometric properties and resistance of plague microbe cultures to technological factors in the process of their concentration using optimized technology did not reveal any significant differences as compared to the routine one. Technological stage of concentrating has been reduced to 5 h term with a three-fold increase in the yield of finished product.

Whole-Genome Assembly of Yersinia pestis 231, the Russian Reference Strain for Testing Plague Vaccine Protection

ABSTRACT We report the whole-genome sequence of Yersinia pestis subsp. pestis bv. Antiqua strain 231 belonging to the 0.ANT3 phylogroup, the reference strain for testing plague vaccine protection in Russia. Genome sequencing was completed using the Oxford Nanopore MinION and Illumina platforms.